Pak. J. Bot., 49(1): 237-247, 2017.
VARIABILITY IN THE GROWTH AND NODULATION OF SOYBEAN IN RESPONSE TO ELEVATION AND SOIL PROPERTIES IN THE HIMALAYAN REGION OF KASHMIR-PAKISTAN NASIR RAHIM1, M. KALEEM ABBASI1* AND SOHAIL HAMEED2 1
Department of Soil and Environmental Sciences, Faculty of Agriculture, The University of Poonch, Rawalakot Azad Jammu and Kashmir, Pakistan 2 Nuclear Institute of Biology and Genetic Engineering (NIBGE), Faisalabad, Pakistan * Correspondence author’s email:
[email protected] Tel.: +92 (0) 5824 960041, Fax: +92 (0) 5824 960004 Abstract
This study was conducted to examine the variability of soybean nodulation and growth in relation to elevation and soil properties across the slopping uplands of the Himalayan region of Rawalakot Azad Jammu and Kashmir (AJK), Pakistan in order to find efficient native N2 fixing bacteria adapted to local soil and climatic characteristics. Soils from twenty two different sites with variable altitude were collected and analyzed for different physico-chemical characteristics including the quantitative estimation of rhizobium population through most probable number (MPN) technique. Soybean cultivar William-82 was grown in these soils under greenhouse condition for determining the nodulation potential (number and mass) and plant growth characteristics. Morphology of the nodules were observed through optical and transmission electron microscopy. Principal component analysis (PCA) and Biplot graph were used to jointly interpret the relationship between variables and soils (treatments). Soil altitude ranged from 855 m to 3000 m while organic matter content varied between 0.8% to 3.5% and pH from 6.0 to 8.1. The indigenous rhizobia population varied between 5.0 x10 4 to 8.0 x106 CFU g-1 showing the existence of a substantial rhizobial population in these soils. The number of nodules per plant varied from 7 to 40 (CV 38%) suggesting site/location as an important factor contributing towards rhizobia population and impacting root nodulation. The electron microscopy of green plant nodules showed densely populated bacteria in these cells and nodule tissue cells were completely infected with bacteria. The growth characteristics of soybean i.e. shoot length, shoot fresh and dry weight, root length, root fresh and dry weight varied among the sites but in general a vigorous and healthy plant growth was observed reflecting N assimilation from native soils. Results showed a substantial variability between sites and this is likely to be due to inter/intra species diversity, as well as changes in microbial community composition/structure. The study suggests that soybean could be planted in this region without prior inoculation, and that native N 2-fixing bacteria might be isolated from these soils and used as biofertilizers (or inoculants).
Key words: Bio-fertilizer, Glycine max, Isolation, Indigenous rhizobia, Nodulation, Principal component analysis. Introduction Introduction and exploitation of soybean [Glycine max (L.) Merr.] in our cropping systems could be a promising management strategy in many aspects. Firstly, soybean is an important oilseed crop with high nutritional value. Soybean protein contains essential amino acids and its oil contains 85% unsaturated fatty acids and is free of cholesterol so it is highly desirable for human consumption (Raei et al., 2008).It is also a source of carbohydrates, minerals (Ca and P) and vitamins A, B and D (Hymowitz et al., 1998; Abbasi et al., 2012). The composition of soybean, as well as the ease and geographical range of its agricultural production, makes it an inexpensive source of oil and protein used as food and animal feed. Secondly, soybean plays an important role in the global and agricultural N cycles by facilitating biological N2 fixation (BNF) into plant-available N. Soybean has been shown to fix an average of 175 kg N ha–1yr–1 in irrigated production, and 100 kg N ha–1 y–1 in dry land production (Unkovich & Pate, 2000; Furseth et al., 2012). Inoculation of soybean seeds with highly effective rhizobia is a common practice in agricultural production, but the success of this technology requires survival and establishment of the inoculated rhizobia in the soil environment (Da & Deng, 2003). Similarly, Wani et al. (1995) stated that despite the considerable capacity for acquiring N from BNF, the inoculation of soybean with rhizobial strains does not necessarily result in yield increase. Soybean can also nodulate freely with native
rhizobia strains and is able to fulfil its N requirement through BNF once the plants are established (Okogun et al., 2004; Singh et al., 2003). Many soils contain an appreciable number of indigenous rhizobia which may or not be effective for BNF despite competitive adaptability to their local environment (Zahran, 1999). Soybean cultivation in many parts of India has shown existence of native rhizobia besides other exotically adapted strains (Sharma et al., 2010). However, selection of niche-based new elite strains adapted to local environmental conditions and to newly bred plant lines is in its infancy (Appunu et al., 2008). The potential for improving BNF through rhizobium inoculation requires knowledge of the abundance and effectiveness of the indigenous rhizobial populations in the soil. The success of soybean crop depends among other factors on the availability of efficient bradrhizobia in soil. The population of rhizobia is reported virtually absent or very low in many zones of Pakistan and in some cases nodulation was completely absent in soybean roots (Aslam et al., 2000; Achakzai et al., 2002). Javid & Mahmood (2010) conducted a field experiment on soybean in a loamy textured soil at Lahore Pakistan and found a maximum of 8-10 nodules in plant roots. However, a substantial nodulation of soybean (6–31 per plant) was reported in the northeast of Pakistan (Khyber Pakhtunkhwa , KPK) where three indigenous land races and two improved varieties were tested at different sowing timings (Muhammad et al., 2012).
NASIR RAHIM ET AL.,
238
The objective of this study was to determine the variability of selected soils/sites characteristics and to evaluate the relative influences of these characteristics on the occurrence of indigenous rhizobial population, nodulation and growth potential of soybean in soils collected from variable altitude from sub-division Rawalakot Azad Jammu and Kashmir, Pakistan. The study will help is to assess whether native N2-fixing bacteria will effectively colonize and perform with planted soybeans without the need for prior inoculation. Materials and Methods Soil sampling and processing: Twenty two different sites from sub-division (Tehsil) Rawalakot Azad Jammu & Kashmir were selected from undisturbed native grassland on the basis of spatial and temporal variation in the year 2012. Fig. 1 shows the geographical location of the study sites. Rough meadow grass (Poatrivialis L.), /Bermuda grass (Cynodondactylon L.) and orchard grass (Dactylisglomerata L.) dominated the vegetation community at all sites. Within each site, soil samples from the surface 0–20 cm were collected from five points by soil auger and mixed as one composite sample. Before sampling, grass, forest litter or any other material on the soil surface were removed. All the samples were taken from topographically similar locations which were approximately within 100 meter from each other. After being brought into the laboratory, soil samples were airdried for 2-3 days, partially milled and subsequently sieved. Samples were lightly ground and subsequently sieved through a 2 mm mesh to remove stones, roots and large organic residues. About one kg soil was taken, sealed in the plastic bags and stored in a refrigerator at 4 0 C prior to analysis. Detail meteorological data of selected sites were also recorded during sampling (Table 1).
Sampling sites Baikh Parat PothiBala HurnaMera Paniola Tain Thorar Dar Rawalakot Tarar Chak Hussainkot Banjosa Jandali Dothan Namnota Khukot Ali Sojal KhyusaneBaik Tolipir Topa Rehara
Laboratory analysis: Sub-samples (triplicate) from each site were taken and analyzed for soil texture, organic matter, total N and most probable number (MPN). Soil texture was determined after the organic matter was removed with 20% H2O2, by the Bouyoucos hydrometer method (Bouyoucos, 1962).Soil pH was determined in distilled water with a glass electrode (soil/H2O ratio 1:2.5 w/v). Soil organic matter (SOM) was determined using a modified Mebius method (Nelson & Sommers, 1982). Briefly, 0.5 g soil was digested with 5 ml of 1N K 2Cr2O7 and 10 ml of H2SO4 at 150 0C for 30 min, digests were then titrated with standardized FeSO4. Total N was determined by the Kjeldahl’s digestion, distillation and titration method (Bremmer & Mulvaney, 1982). The rhizobial population in the soil samples was determined by serial dilution and colony forming unit (CFU) on YEM agar plates by spread plate method (Somasegran & Hoben, 1994). Briefly, one gram of soil from each sample was added to 9 mL of 0.89 % saline solution and serial dilutions were made. From each dilution (10-5, 10-6, 10-7) 100 µL was spread using sterile glass spreader on YEM agar plates. The plates were incubated at 28oC for 3 to 5 days and CFU was counted. Soybean growth under greenhouse conditions: A pot experiment was conducted to assess soybean growth and nodulation in soils collected from each of the 22 sites. Six soybean seeds (cultivar William-82) were planted in pots (18 cm width x 17 cm height) in May 18, 2012. The pots were arranged in a randomized complete block (RCB) design. After germination, the pots were thinned to keep only the 3 healthiest seedlings. No fertilizer was applied in any pot, however, irrigation was given when needed. Pots were kept in the greenhouse of the Faculty of Agriculture, Rawalakot.
Table 1. Meteorological data of the selected sites for the year 2012. Altitude (m) Air Temp. (oC) Soil Temp. (oC) 1747 30.8 22 1379 32.5 24 1849 27.5 22 2037 30.5 22 1341 33.0 23 855 31.0 30 1646 26.0 23 1090 29.4 29 1594 29.0 27 1812 27.6 22 1647 28.0 25 1818 26.0 22.5 1779 26.0 23.0 2026 24.0 22.5 1774 25.0 23.0 1763 26.2 24.0 1631 27.5 24.0 1700 28.0 24.0 2274 23.0 22.0 3000 22.0 20.0 2008 24.6 22.0 1544 28.5 26.0
Humidity (%) 62.2 59.2 65.4 57.2 58.0 53.8 73.8 66.5 55.4 48.5 49.5 56.0 73.0 73.0 68.0 66.0 59.8 58.4 71.0 75.0 53.3 68.0
VARIABILITY IN GROWTH AND NODULATION POTENTIAL OF SOYBEAN
239
Fig. 1. Geographical representation of the state of Azad Jammu and Kashmir (a) and points on right side (b) representing the sampling sites located at Rawalakot Azad Jammu & Kashmir.
At harvest, plants were carefully taken out of the soil to avoid root damage. Roots were then separated from the shoots and all adhering soil particles were washed away with water. The nodules were separated from the roots, counted and weighted fresh. Plant shots, roots and a subsample of nodules were oven-dried at 70°C for 48 hours to measure dry weight.
the section of 120-200 nm thickness. The sections were mounted on copper grids and stained with uranyl acetate for 30 minutes and lead acetate for 10 minutes. After staining the grids were washed with distilled water. The observations of samples were taken at light microscope (LEICA DM LS) and transmission electron microscope JEOL JEM-1010 (Hameed, 2003).
Optical and transmission electron microscopy: Bacterial occupancy in the root nodules was examined using light and transmission electron microscope. Nodules samples were collected from soybean plants at flowering. Nodules were cut into 1-3 cm pieces and embedded in water agar to cut 2-3 mm small cubes. These small cubes were fixed in 5% gluteraldehyde for 16-18 h. The samples were then washed two times in 0.2 M PIPES buffer and treated with 0.2% osmimiumtetraoxide prepared in 0.2 M PIPES buffer (pH6.8) for 16-18 h. After washing samples were treated with 5% aqueous uranyl acetate for 16-18 h and again washed with sterilized distilled water. After dehydration with absolute alcohol and then with 100% propylene oxide, samples were transferred to flat embedding moulds containing pure resin and polymerized at 65oC for 72 h. After polymerization the resin blocks were left at room temperature for at least 24 h before sectioning. Sectioning was performed at ultra-microtone (RMC-7000) to slice
Principle component analysis: To find out variation in growth and nodulation potential of different soils, principle component analysis (PCA) was done. Eighteen principle components (PCs) were extracted. The Jolliffe cut-off value was 0.7 was used as an indication of significant principal components (Jolliffe, 1986). Statistical analysis: One way analyses of variance was performed using Anon., (1991) statistical analysis package to determine sites effect on the nodulation and growth components of soybean. All statistical comparisons were made at α = 0.05 probability level using the least significant difference method for mean separation (Muhammad, 1995).Average values of each trait were analyzed for multivariate analysis (principal component analysis) by using the SPSS-12 for Windows (www. SPSS. com) as described earlier (Iqbal et al., 2008).Correlation and linear analyses were used to examine the relationship among different variables by using SPSS 12 for Windows (www. SPSS.com).
240
Results Locations and sites characteristics: Soil samples collected from twenty two sites of the same ecological zone of Rawalakot Azad Jammu and Kashmir had shown a wide variation in altitude ranged between a minimum of 855 m at Tain to a maximum of 3000 m at Tolipir site (Table 1). Soil temperature of these soils at the time of sampling was between 220C0 to 300C while the humidity of the selected sites varied between 48.5 % to 75 % maximum at Tolipir and minimum at Trar site. A significant negative correlation existed between altitude and air temperature (r=–0.74) and between altitude and soil temperature (r=–0.81) while the correlation between altitude and humidity was non-significant (r=0.38) (Table 2).Coefficients of variation (CV%) for elevation was high (24%) reflecting the rolling topography typical of this resource area while temperature and humidity showed less variability (10 and 13%) showing that both traits were relatively stable in this ecological zone. Variability in soil physico-chemical characteristics: Data regarding the physico-chemical characteristics of different sites are presented in Table 3. These soils varied in texture, seven soils are loam, six soils are sandy loam, five soils have silt loam texture, three soils have silty clay loam texture and one soil belong to the class sandy clay. The clay content of these sites varied between 14–35%, sand and silt 15–55%. The pH of the soils varied from 5.89 – 8.08 showing acidic to alkaline nature of the soils. Out of 22 soils, two soils having pH 6 or below (acidic), eleven soils pH between 6.5 -7.5 (neutral), three soils pH .8.0 and six soils pH >7.5 and
